The invention relates to a method for suppressing angiotensin-converting enzyme (ACE), a composition effective for this purpose and a method for preparing the composition, specifically by enzymatic conversion of whey proteins.
Hypertension has been reported to be the most important cause of human deaths in industrialized countries. (See, for example, Laragh, J. H., 1979. L'hypertension. Recherche, 105 (10): 1068–1076.) Nearly 30% of the fatalities among adults would result from hypertension or from its renal, coronary or neurological complications. The elucidation of the physiological mechanisms responsible for hypertension has lead the pharmaceutical industry to propose angiotensin converting enzyme (ACE)—inhibitory substances. ACE catalyses the degradation of angiotensin I into angiotensn II, a strong vasoconstrictor.
Peptides found in Brazilian snake venom have been identified as the most effective natural substance for the inhibition of ACE. (See, Ferreira, S. H., Bartelt, D.C., Greene, L. J., 1970. Isolation of bradykinin-potentiating peptides from Bothrops jararaca venom. Biochemistry, 9 (13): 2583–2593.) The inhibitory effect of natural peptides has been related to their binding at the active site of ACE. (See, Maubois, J. L., Léonil, J., Trouvé, R. Bouhallab, S., 1991. Les peptides du lait á activité physiologique III. Peptides du lait á effet cardiovasculaire: activités antithrombotique et antihypertensive. Lait, 71: 249–255.)
A structure-function study of these various bioactive peptides has suggested that they often possess a Pro-Pro, Ala-Pro or Ala-Hyp at their C-teminal sequence. (See, Maruyama, S., Suzuki, H., 1982. A peptide inhibitor of angiotensin I converting enzyme in the tryptic hydrolysate of casein. Agric. Biol. Chem., 46 (5): 1393–1394; and Oshima, G., Shimabukuro, H., Nagasawa, K. 1979. Peptide inhibitors of angiotensin I-converting enzyme in digests of gelatin by bacterial collagenase. Biochim. Biophys. Acta, 566: 128–137.) The occurrence of proline might also contribute to the ACE-inhibitory activity of peptides derived from food proteins. (See, Kohmura, M., Nio, N., Kubo, K., Minoshima, Y., Munekata, E., Ariyoshi, Y. 1989. Inhibition of angiotensin-converting enzyme by synthetic peptides of human β-casein. Agric. Biol. Chem., 53 (8): 2107–2114.)
Maruyama and Suzuki [supra] have evidenced such amino acid sequences in peptides from tryptic casein hydrolysates. The authors have shown that the peptide f23–34 from xsl casein (bovine, variant B), possesses ACE-inhibitory activity estimated by an IC50 value (concentration needed to inhibit 50% ACE activity) of 77 μM. Numerous other studies followed this work and revealed other ACE-inhibitory peptides in casein hydrolysates. In a recent review, Nakano has reported the occurence of 18 distinct milk protein-derived peptide sequences, found in sour milk, and which have been shown to possess ACE-inhibitory activity. (Nakano, T., 1998, Milk derived peptides and hypertension reduction. Int. Dairy J., 8: 375–381.)
However, only a few studies have reported the occurrence of ACE-inhibitory activities among whey proteins hydrolysates. Abubakar, et al., have determined the ACE-inhibitory activity in whey protein hydrolysates using seven different enzymes: trypsin, proteinase-K, actinase-E, thermolysin, papain, pepsin and chymotrypsin. It was shown that the specificity of the enzyme had a pronounced effect on the resulting ACE-inhibitory activity of the hydrolysate, and that the biological activity was originating from the major whey proteins (β-1g, α-1a, BSA, Ig) and not from the caseinomacropeptide. (Abubakar, A., Saito, T., Aimar, M. V., Itoh, T. 1996. New derivation of the inhibitory activity against angiotensin converting enzyme (ACE) from sweet cheese whey. Tohoku J. Agric. Res., 47 (1–2): 1–8.) More recent work from. Abubakar, et al., has allowed the identification of nine peptide sequences, namely β2-microglobulin (f18–20), β-lactoglobulin (f78–80), serum albumin (f221–222), β-casein (f∇–61, f59–64, f62–63, f80–90, f157–158, f205–206), among which β-lactoglobulin (f78–80) showed the strongest antihypertensive activity in spontaneously hypertensive rats. (Abubakar, A., Saito, T., Kitazawa, H., Kawai, Y., Itoh, T., 1998, Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion. J Dairy Sci., 12: 3131–3138.) Finally, Mullaly et al., have demonstrated that a peptidic fraction, isolated by using RP-HPLC, from a tryptic hydrolysate prepared with bovine β-lactoglobulin had an IC50 value of 159.8 μmol/L, compared to Captopril, a commercial drug commonly used in hypertension treatment, which has an IC50 of 0,006 μmol/L. (Mullally, M. M., Meisel, H., FitzGerald, R. J., 1997. Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine β-lactoglobulin. FEBS Letters, 402: 99–101.) Mass spectrometry analyses have allowed the identification of peptide f142–148 from β-lactoglobulin as being responsible for the ACE-inhibitory activity in tryptic hydrolysates of β-lactoglobulin. The same peptidic sequence obtained by chemical synthesis showed an IC50 of 42.6 μmol/L.